Method to treat endotoxin effects by administration of 33 kilodalton phospholipid binding protein

ABSTRACT

Pharmaceutical compositions containing safe and effective amounts of Annexin I or 33 kDa(PLBP) can be used in a number of methods for the benefit of animals.

This invention was made with United States government support awarded byNIH, Grant # NIH Project #'s. HL 38744 and P50 HL46478. The UnitedStates Government has certain rights in this invention.

FIELD OF THE INVENTION

The present invention generally relates to annexins. More particularly,it relates to pharmaceutical compositions containing an annexin andmethods of treating lung disease and endotoxin shock in animals,including humans.

BACKGROUND OF THE INVENTION

Annexins are a group of calcium-dependent, phospholipid-bindingproteins. The calcium and phospholipid binding sites of most annexinsare located in four repeated and highly conserved regions each of whichcontains about 70 amino acids. These proteins are widely distributed andat least nine members of the annexin family of proteins have beenidentified in mammalian tissues.

The lung is rich in annexins. Several members of the annexin family ofproteins with apparent molecular weights ranging from about 32 to about40 kDa have been isolated from lungs of animals. Annexin I, a 36 kDphospholipid binding protein, 36 kDa(PLBP), appears to be the mostabundant of the annexin family of proteins in the lung. There is aboutfive times more Annexin I present in the lung than the related annexin33 kDa phospholipid-binding protein, 33 kDa(PLBP).

Compared to the whole lung, the alveolar epithelial type II cells inwhich the pulmonary surfactant complex is synthesized, stored andsecreted have higher expression levels of Annexin I. In addition to theintracellular localization of Annexin I in alveolar type II cells, thisprotein has been found in lung lavage fluids from human and animals. Alikely source of the Annexin I in the lung lavage fluid is the alveolartype II cells.

Using an antibody to Annexin I, researchers have found Annexin I and asmaller protein in the bronchoalveolar lavage (BAL) fluid of patientswith lung diseases. The smaller protein was found to be a proteolyticbreakdown product resulting from the action of neutrophil elastase uponAnnexin I in the patients' BAL fluid. The discovery of the Annexin Ibreakdown product in human BAL fluid samples is consistent with thereport that the Annexin I N-terminal region is subject to cleavage byvarious proteases to yield breakdown products.

I have discovered that a breakdown product of Annexin I, which has amolecular weight of about 33 kDa (33 kDa(BP)), immunoreacts withanti-Annexin I antibodies. I also have discovered that 33 kDa(BP) iscytotoxic and that it is present in higher concentrations in the BALfluid of patients with lung diseases, including cystic fibrosis (CF),and premature infants with chronic lung disease (bronchopulmonarydysplasia (BPD) than in normal humans. In addition, the BAL fluid ofpatients with lung disease contains less Annexin I than that of normalhumans. Based on these and other discoveries, I have found thatadministering Annexin I or 33 kDa(PLBP) to lung disease patients can bebeneficial to such patients. I also have made the further discovery thatthe administration of Annexin I or 33 kDa(PLBP) can be beneficial in thetreatment of endotoxin toxicity and inflammation. The major pathogenesisof endotoxin toxicity leads to inflammation and septic shock.

Bacteria or bacterial products, such as endotoxin from gram-negativebacteria, activate host response during infectious and inflammatoryprocesses. Endotoxin, also known as lipopolysaccharide (LPS) for itschemical structure consisting of a polysaccharide part and a hydrophobiclipid part, can induce a wide variety of different types of cellsincluding macrophages, polymorphonuclear leukocytes, and endothelialcells to release a number of inflammatory mediators, such asprostaglandins or cytokines. In localized infections, endotoxin islargely restricted to inflammatory sites, enhancing host defense.However, if the infection is not brought under control, endotoxin and/orinflammatory mediators may reach the circulation, predisposing themicrovasculature to thrombosis and can lead to systemic endotoxemia orsepsis and associated complications including septic shock, adultrespiratory distress syndrome, and multiorgan failure.

Septic or endotoxin shock is an acute and serious cardiovascularcollapse resulting from the systemic response to a bacterial infection.It is manifested by hypotension, a reduced response to vasoconstrictors,generalized tissue damage and multi-organ failure. It is the most commoncause of death in the intensive-care unit; there are about 400,000 casesof septicemia per year in the United States with mortality rates between25% and 50%. The steadily increasing incidence of septic shock stemsfrom an increasing proportion of elderly in the population, increasingfrequency of invasive surgical procedures, extensive use ofimmunosuppressive and chemotherapeutic agents, and increasing prevalenceof chronic debilitating conditions. Because the mechanisms underlyingsepsis and septic shock are not yet known, therapeutic interventionshave been largely ineffective. At present, there is no effectivetreatment for septic shock.

It would be advantageous both to have methods of treating lung diseases,including cystic fibrosis (CF) and bronchopulmonary dysplasia (BPD), andtreating endotoxin shock.

SUMMARY OF THE INVENTION

It is an object of the present invention to disclose novelpharmaceutical compositions containing annexins that can be used inmethods of treating lung disease patients or in methods for treatingpatients with endotoxin shock.

The pharmaceutical compositions of the present invention comprise amember selected from Annexin I and 33 kDa(PLBP) and mixtures of thosetwo polypeptides, in combination with pharmaceutical diluent(s) andexcipients. The preferred compositions also contain a source of calciumions (Ca⁺²). Especially preferred for treating lung disease or endotoxinshock in animals are compositions which are suitable for instillationinto the bronchial system of an animal.

In one method of the present invention, a composition containing amember selected from the group consisting of Annexin I, the 33 kDa(PLBP)and mixtures thereof, is administered to an animal having a lung diseasein an amount which is safe and effective to improve the conditions ofthe lungs of said animal.

In another method of the present invention, a composition containing amember selected from the group consisting of Annexin I, the 33 kDa(PLBP)and mixtures thereof, is administered to an animal in an amount which issafe and effective to prevent or alleviate the adverse effects ofendotoxin in said animal.

These and other objects of the invention will be apparent from thedescription and examples herein.

DESCRIPTION OF THE PREFERRED EMBODIMENT

The preferred pharmaceutical compositions are those which contain as theactive ingredient the 33 kDa(PLBP). The reason that the 33 kDa(PLBP) ispreferred is that it is as active as Annexin I both in the counteractingof the effects of endotoxin and in the treatment of lung disease and itis less likely to be broken down into cytotoxic products by enzymes.

In addition to the active ingredient and a source of calcium ions, thepreferred compositions for instillation into the airway of an animalwill contain a surfactant.

In the preferred methods of treating lung disease and preventing theadverse effects of endotoxin, the compositions are instilled directlyinto the patient's lungs. Therefore, the compositions also may containpharmaceutical diluents and excipients which are customary for aerosolsor other dosage forms for instillation of drugs into the airways ofanimals.

The practice of the present invention will be further understood by thedescription of the experimental work that follows.

Experimental Work I. Degradation of Annexin I in Lung Disease

A study was conducted to determine whether degradation of Annexin Ioccurs in BAL fluid of patients with lung disease, such as cysticfibrosis (CF) and bronchopulmonary dysplasia (BPD). The lung disease ofCF is characterized by bacterial infection and inflammation. As aresult, the patient's mucus contains high amounts of proteases,particularly, the elastase from the neutrophils and from the organismPseudomonas aeruginosa that colonized the respiratory tract of the CFpatients. It was speculated that these proteases may break downproteins, including annexins, in the bronchi and bronchiole. The lungdisease of premature infant BPD is characterized by oxyradical-mediatedacute lung inflammation and injury. These premature infants survivedrespiratory distress syndrome (RDS) after intensive care but sufferedoxygen toxicity and developed bronchopulmonary pulmonary dysplasia, achronic lung disease. The BAL fluid of these patients also containedhigh level of proteases derived from neutrophils in addition to theoxyradical substances. In this study, rabbit lung Annexin I, which isequivalent to human Annexin I, was used and specific antibodies wereraised against rabbit lung Annexin I to analyze the distribution ofAnnexin I in BAL fluid from CF patients and BPD patients and todetermine the changes in Annexin I structure and functional activity.

Isolation of lung annexins. Rabbit lung Annexin I was isolated from thecytosolic fraction of the lungs from two adult rabbits by knowntechniques. Human lung Annexin I was isolated from post-mortem lungtissue.

Isolation of lung annexins and preparation of anti-Annexin I antiserum.The two rabbit lung calcium-dependent phospholipid-binding proteins,Annexin I or 36 kDa(PLBP) and 33 kDa(PLBP), were isolated from cytosolicfraction of the lungs from adult rabbit by known techniques (Tsao FHC,Biochimica et Biophysica Acta. 1045 (1990) 29-39, incorporated byreference.) The 36 kDa(PLBP) was identified to be rabbit Annexin I (TsaoFHC, et al. Biochimica et Biophysica Acta. 1081 (1991) 141-150). Humanlung Annexin I was isolated from post-mortem lung tissue by the sametechniques. The purified rabbit lung Annexin I was used as antigen toraise specific antibodies in guinea pigs. The guinea pig antiserum torabbit lung Annexin I (gpAb-Anx-I) was highly specific for rabbit lungAnnexin I and cross-reacted with human Annexin I (Tsao FHC et al.Biochimica et Biophysica Acta. 1213 (1994) 91-99).

Analysis of annexin in bronchoalveolar lavage (BAL) fluid samples.Bronchoalveolar lavage fluid was obtained from normal volunteersubjects, patients with CF, patients with interstitial lung disease(ILD), and patients with bronchopulmonary dysplasia (BPD). The BALfluids were concentrated 5 to 10-fold by centrifugation (3000×g at 4°C.) using Amicon Centricon10 filters (molecular weight cut-off of 10kDa) (Beverly, Mass.). Fluid retained by the filter was saved forfurther analysis. An aliquot of BAL fluid sample containing 0.1 mg oftotal proteins was lyophilized in Speed Vac (Savant Instruments, Inc.,Farmingdale, N.Y.) to dryness. The sample proteins were resuspended in20 μl of sample buffer containing sodium dodecyl sulfate (SDS) anddenatured in boiling water for 5 min. The proteins were separated by SDSpolyacrylamide gel electrophoresis (SDS-PAGE) under the denaturedconditions using a vertical 10% SDS gel (7×8 cm). Proteins on the SDSgel were then electrophoretically transferred onto a nitrocellulosemembrane. Annexins on the membrane were immunoblotted by the polyclonalantibody raised in guinea pig against rabbit lung Annexin I(gpAb-anx-I). As specified, in some studies proteins on the SDS gel werevisualized by silver staining (Silver Staining Kit, Sigma Chemical Co,St. Louis, Mo.). The isoelectric point (pI) values of annexins in theBAL fluid samples with 50 μg of total proteins were determined byisoelectric focusing (IEF) using an IEF agarose gel with pH rangebetween 3 and 10 (10×12.5 cm, FMC, Rockland, Me.). Proteins on the IEFgel were transferred to a nitrocellulose membrane by capillary force,and annexins on the membrane were analyzed by Western blot usinggpAb-anx-I.

Effects of BAL fluid from CF patients on the activity and structure ofAnnexin I. An amount of purified rabbit lung Annexin I was incubatedwith CF BAL fluid samples in a ratio of 1 μg Annexin I/20 μg BAL proteinin 10 μl of 0.01M Tris-HCl, pH 7.4 (for Western blot as describedabove), or in a ratio of 10 μg Annexin I/100 μg BAL protein in 50 μl of0.01M Tris-HCl, pH 7.4 (for Annexin I activity measurement), at 37° C.for 1 h. The Annexin I activity was determined by measuring theaggregation of ¹⁴ C-labeled phosphatidylcholine unilamellar liposomes tomultilamellar liposomes by known techniques.

In a separate experiment, an amount of 0.2 mg of purified rabbit lungAnnexin I was incubated with BAL fluid containing 0.42 mg total proteinsin 0.3 ml of 0.01M Tris-HCl, pH 7.4, at 37° C. for 2 h. After reaction,the reaction mixture was centrifuged at 100,000×g for 10 min. Annexin Iin the supernatant was isolated by HPLC C4 Vydac reverse phase asdescribed above. The purity and the molecular weight of Annexin Iobtained from HPLC reverse phase column were examined by SDS-PAGE andWestern blot. The N-terminal sequence of Annexin I from HPLC reversephase was determined using an automated Model 477A Liquid PulseSequencer and Model 475A Gas Phase Sequencer with on-line Model 120A PTHAnalyzer and 610A Data Analysis System (Applied Biosystems, Foster City,Calif.).

Western blot analysis of annexins in human BAL fluid. With the use of100 μg total BAL fluid proteins, Annexin I was detected in BAL fluidsamples from normal volunteers by Western blot. Annexin I in one of the10 normal volunteer BAL samples was only barely detected. Annexin I alsowas present in all 12 BAL fluid samples from patients with interstitiallung diseases. Small amounts of an immunoreacted protein with molecularweight around 33 kDa also was observed in some of the samples. This 33kDa protein did not bind with phospholipid and was determined to be the33 kDa(BP) breakdown product of Annexin I. It appeared that the BALfluid samples of patients with interstitial lung disease which had about20% neutrophil among total BAL fluid cells also contained small amountsof the 33 kDa(BP). In contrast, in 20 BAL fluid samples from CFpatients, 17 samples had no Annexin I. In 11 of the 17 samples with noAnnexin I, the only immunoreactive protein was the 33 kDa(BP). The other6 among the 17 BAL fluid samples had no detectable immunoreactiveproteins at all. Among the 20 CF BAL specimens, only three samples hadAnnexin I, but two of these three samples also contained theimmunoreactive 33 kDa(BP). Interestingly, the three CF BAL samples whichcontained Annexin I also had lower neutrophil elastase activities. Allthe BAL fluids of CF patients contained neutrophils in concentrationsover 10⁵ cells/ml, compared to the very low concentrations ofneutrophils in normal volunteers and patients with interstitial lungdiseases. The additional two BAL fluid samples from normal volunteersalso contained Annexin I, but one of these two samples also containedthe 33 kDa(BP).

Conversion of Annexin I to 33 kDa(BP) by CF BAL fluid and elastase. Theincubation of purified rabbit lung Annexin I (1 μg) with four differentCF BAL fluid samples (20 μg protein) yielded 33 kDa(BP) which wasimmunorecognized by the antibody. The 33 kDa(BP) was solely derived fromsubstrate of rabbit lung Annexin I since the four CF BAL samplesemployed in the tests contained only 20 μg of total proteins in whichlittle Annexin I and 33 kDa(BP) could be detected. Under the reactionconditions, three BAL fluid samples converted most of the Annexin I tothe 33 kDa(BP), whereas one BAL fluid sample degraded less Annexin I tothe 33 kDa(BP). Interestingly, that CF BAL fluid sample also containedboth endogenous Annexin I and 33 kDa(BP), whereas the other three CF BALsamples used in the reactions had no Annexin I but only the 33 kDa(BP).

The Annexin I breakdown product, the 33 kDa(BP), generated by a CF BALfluid sample had a basic isoelectric point value with pI at 8.5, whichwas markedly different from the pI of 6.0 of Annexin I or the pI of 5.5of 33 kDa(PLBP). The M_(R) values of Annexin I and 33 kDa(PLBP) andtheir structures were determined as described in the literature.

The incubation of purified rabbit lung Annexin I with CF BAL fluidsamples in which Annexin I was absent resulted in a decrease in AnnexinI activity in liposome aggregation. Contrarily, the incubation of rabbitlung Annexin I with a CF BAL sample in which endogenous Annexin I waspresent did not affect Annexin I activity.

Elastase also degraded rabbit lung Annexin I into the 33 kDa(BP) whichwas immunorecognized by gpAb-anx-I. The presence of phenylmethylsulfonyl fluoride (PMSF) in the reaction solution totally inhibited theproteolytic hydrolysis of Annexin I catalyzed by elastase.

In the experimental work described Annexin I was present in all the BALfluid samples from normal volunteers. The finding of Annexin I in BALfluid from normal volunteers was consistent with the previous reportsthat Annexin I was present in lung lavage fluid from animals and humans.Little degradation of Annexin I was observed in the BAL fluid samplesfrom normal volunteers. However, degradation of Annexin I appeared to becommon in all the BAL fluid samples from CF patients. In most of the CFsamples, Annexin I was completely degraded to 33 kDa(BP). Only a few CFsamples contained any Annexin I, but even those samples also had the 33kDa(BP) protein. Although Annexin I was present in all the BAL fluidsamples from patients with interstitial lung diseases, some of thesamples also contained the 33 kDa(BP) protein.

It is interesting to note that among the BAL fluid samples from patientswith interstitial lung diseases, the appearance of 33 kDa(BP) wasassociated closely with relatively higher percentage of neutrophils inthese samples. Since all the BAL fluid samples from CF patientscontained abundant neutrophils, it was concluded that the degradation ofAnnexin I to the 33 kDa(BP) was associated with neutrophils. Likely, thehigher the neutrophil elastase in the BAL fluid, the more degradation ofAnnexin I took place. For those CF BAL fluid samples which had lowelastase activity, Annexin I was present in the BAL fluid. Thus, thebreakdown of Annexin I in BAL fluid was associated closely with thedegree of lung inflammation in the CF patients.

The proteolytic activity in the CF patients' BAL fluid further wasconfirmed by the degradation of purified rabbit lung Annexin I incubatedin reaction mixtures containing CF BAL fluid. The breakdown product 33kDa(BP) of rabbit lung Annexin I catalyzed by CF BAL fluid structurallywas nearly identical to the human lung Annexin I breakdown product inthe CF BAL fluid samples, i.e., same molecular weight and pI between8.5-9.0. The Annexin I breakdown product 33 kDa(BP) protein had a pIvalue which was distinct from the lung 33 kDa(PLBP), which was an acidicpI of 5.5. Both rabbit lung Annexin I and 33 kDa(PLBP) aggregatednegatively charged vesicles in a calcium-dependent manner, an importantannexin functional activity. The degradation of rabbit lung Annexin Icatalyzed by CF BAL fluid markedly reduced the Annexin I functionalactivity, in other words, the Annexin I breakdown product 33 kDa(BP) wasfunctionally inactive in vesicle aggregation.

Although previous studies suggested that the degradation of Annexin I inBAL fluid from patients with lung diseases was due to the elastasehydrolytic activity, we found that the cleavage site of Annexin I was atthe N-terminus Val-36, an elastase substrate specific cleavage site.Also the cleavage site of Annexin I was determined with rabbit lungAnnexin I which was used as the substrate. Both rabbit lung Annexin Iand human lung Annexin I have nearly identical amino acid sequences inthis region. It also was determined that human lung Annexin I could becleaved at Ser-37. This suggested that degradation of Annexin I couldoccur at more than one position at the N-terminus. It has been shownthat the N-terminus of Annexin I can be cleaved at several positions bydifferent proteases, such as cathepsin D, calpain or plasmin, which havebeen demonstrated to cleave human annexin I at Trp-12, Lys-26 or Lys-29,respectively. The N-terminus truncated Annexin I has been shown toeither increase or decrease the binding affinity with calcium andphospholipid, depending on the N-terminus-truncated position. It hasbeen demonstrated that the removal of 36 amino acids at the N-terminusof Annexin I by the proteases in CF patients' BAL fluid diminished theAnnexin I functional activity in vesicle aggregation and fusion,indicating that the N-terminus is required for Annexin I functionalactivity.

The source(s) of Annexin I in BAL fluid are not known with certainty. Ithas been found that alveolar epithelial type II cells are rich inAnnexin I. Though the role of Annexin I in the type II cell is notclear, it might be associated with lung surfactant and possibly secretedby type II cells. The pulmonary surfactant appears not only to beessential to stabilize alveoli from collapse at the lowest volume duringexpiration, it also may play an important role in mucociliary clearancein the respiratory tract. The abnormal surfactant phospholipidcomposition in the mucus of CF patients not only may contribute to theabnormal mucociliary transport, but it also may cause the collapse ofterminal airways. The degradation of Annexin I in the BAL fluid in CFpatients was not only a sensitive indicator of the high levels ofneutrophils and elastase in the inflammatory lung, it also was anindication of the decreased anti-inflammatory activity due to thereduction of the levels of Annexin I in the lungs of these patients.Similarly, Annexin I in the BAL fluid samples from five BPD patients wasall degraded to 33 kDa(BP). These patients had acute lung inflammationand injury.

From the foregoing, it was apparent that administering Annexin I or 33kDa(PLBP) to patients with lung disease would be beneficial to suchpatients.

II. Isolation and Structural Determination of 33 kDa(BP)

The rabbit lung Annexin I, after incubation of Annexin I with CF BALfluid at 37° C. for 2 h, was purified by HPLC reverse phase column. ThisAnnexin I was eluted as a single peak at the 35-min elution time fromthe column. Some minor proteins, presumably from BAL fluid, were elutedearlier than Annexin I. The apparent molecular weight of this Annexin Iwas determined to be 33 kDa by SDS-PAGE. Thus, this annexin protein wasdesignated as Annexin I breakdown protein, or 33 kDa(BP).

Amino acid sequence determination of 33 kDa(BP) add human lung AnnexinI. The 15 amino acid residues determined for the sequence of the 33kDa(BP) protein derived from rabbit lung Annexin I matched the aminoacid sequence between Ser-37 and Leu-51 of human Annexin I whose entireamino acid sequence had been deduced from cDNA as described in theliterature. Among the 15 amino acids, Thr-41 in human Annexin I sequencewas replaced with Phe in the 33 kDa(BP) protein and Asp-47 in humanAnnexin I sequence could not be determined for the 33 kDa(BP) protein.

The results of Western blot also showed that the immunoreacted Annexin Iin human BAL fluid had the same apparent molecular weight as rabbit lungAnnexin I. The molecular weight of human Annexin I calculated from aminoacid sequence deduced from cDNA is 38712.16. Recently, the rabbitAnnexin I cDNA was cloned and sequenced; the deduced protein sequencehas 346 amino acids with a calculated molecular weight of 38831.28,similar to the Annexin I of human, rat, mouse and guinea pig. Theapparent molecular weight of rabbit lung Annexin I was 36 kDa asvigorously examined by SDS gel. The observed molecular weights of humanAnnexin I also have been reported to be around 35-37 kDa. The differencein molecular weights between the calculated and experimentallydetermined might be the result of protein charge effects on proteinmigration on SDS gel due to some post-translational modifications. To beconsistent with the observed molecular weight, lung Annexin I isreferred to as 36 kDa(PLBP) protein since Annexin I was analyzed mostlyby SDS-PAGE and Western blot.

III. Inhibition of Endotoxin and Cytotoxicity by Annexin I and 33kDa(PLBP)

i. In Vitro Study of Anti-Endotoxin and Anti-Cytotoxicity Activities ofAnnexin I and 33 kDa(PLBP).

I have discovered that the Annexin I and 33 kDa(PLBP) have a highaffinity for binding to endotoxin or lipopolysaccharide (LPS) and thatthe binding is calcium dependent. Both Annexin I and 33 kDa(PLBP)effectively inhibited endotoxin stimulation on macrophage release ofcytokines. Also, Annexin I and 33 kDa(PLBP) inhibited killing of cellsby cytokines released by activated macrophages. Overall, Annexin I and33 kDa(PLBP) prevented endotoxin toxicity.

In the same in vitro cytotoxic assay in which it was demonstrated thatboth Annexin I and the 33 kDa(PLBP) protein prevented the killing ofcells by the breakdown product of Annexin I 33 kDa(BP).

ii. Annexin I and 33 kDa(PLBP) Inhibit Cytotoxicity of BAL Fluid from CFPatients and BPD Patients.

I have found that the BAL fluid from either CF patients or BPD patientskilled cells to a certain extent. These BAL fluid samples contained the33 kDa(BP) but had no Annexin I. I also discovered that when Annexin Ior 33 kDa(PLBP) was added to the cell culture medium, these proteinsprotected cells from killing by the BAL fluid of CF or BPD patients.

iii. In Vivo Study of Annexin I Against Endotoxin.

In this experiment, 6 New Zealand white rabbits (40-days old) wereanesthetized with Ketamine followed by injection of 2 ml of saline intothe tracheas of two control rabbits, 2 ml of saline containing 0.2 mgLPS into two Endotoxin group rabbits, or 2 ml of saline containing 0.2mg LPS and 0.2 mg purified rabbit lung Annexin I into twoEndotoxin+Annexin I group rabbits. Anal temperature of each rabbit wasmeasured every half or one hour after injection.

A tracheal injection of LPS into rabbits induced response within onehour. The animals had body temperatures 2°-3° F. higher than thecontrols within 5 hours. The body temperatures of the rabbits returnedto normal levels 6 and 7 hours after injection. The injection of amixture of LPS-Annexin I-calcium delayed the effects of endotoxin morethan 2 hours and reduced the degree of response. The rabbits thatreceived Annexin I eventually developed fever 4 hours later afterinjection. This is probably due to only one level of Annexin I beingtested versus a large dose of endotoxin and the removal or degradationof Annexin I in the airway and the residual endotoxin causing infection.The difference between the Control and the average of the Endotoxin andEndotoxin+Annexin I groups for all times 3 hours and after was -1.97degrees F. (p-value<0.001) while the difference for these times betweenthe Endotoxin and Endotoxin+Annexin was 0.85 degrees F. (p-value 0.014).This indicates that, after a brief incubation period, theEndotoxin+Annexin I group had a significantly lower average temperaturethan the Endotoxin group.

All the rabbits used in the in vivo study (trachea injection ofendotoxin, Annexin I and endotoxin, or saline) were normal rabbits andthey had endogenous Annexin I. This shows that introducing additionalexogenous Annexin I into the airway of even healthy rabbits can inhibitendotoxin toxicity. The quantity of Annexin I in the airway is importantin protecting against endotoxin. Therefore, introducing exogenousAnnexin I and/or 33 kDa(PLBP) into an animal's airway will enhance theanimal's defense mechanism.

The Annexin I anti-endotoxin activity is probably due to Annexin Ibinding to the lipid-A moiety of LPS so the LPS can no longer bind tothe host cell membrane to trigger cellular reactions that releaseinfectious mediators. Annexin I may also bind to the epithelial cellsurfaces to prevent LPS from anchoring on the cell membrane to initiatecellular reactions. All the results indicated that Annexin I and 33kDa(PLBP) can effectively inactivate endotoxins. Moreover, since thesecompounds are natural products of the lung their administration into theairway of animals, including humans, has minimal or no side effects.

IV. Uses for the Annexin I Breakdown Product, 33 kDa(BP)

33 kDa(BP) is an endogenous cytotoxic substance which can be used incell culture and animal models to study cytotoxicity and apoptosis inlaboratories. Since endogenous 33 kDa(BP) is not easy to obtain, thecommercial production of 33 kDa(BP) will be useful. 33 kDa(BP) can bereadily made in bacteria by the recombinant DNA techniques. Thepost-translational modification of the protein is not a concern insynthesizing 33 kDa(BP) in bacteria since Annexin I charge modificationoccurs at the N-terminus which is depleted in 33 kDa(BP) anyway.

The presence of 33 kDa(BP) in the diseased or inflamed lung appears tobe a critical endogenous apoptosis factor that causes epithelial celldeath and lung injury. Thus, the Annexin I/33 kDa(BP) ratio in BAL fluidcan be used as diagnostic tool to predict lung injury. I have discoveredthat Annexin I and/or 33 kDa(PLBP) can effectively inhibit the cytotoxicactivity of the Annexin I breakdown product 33 kDa (BP) .

The 33 kDa(BP) also can be useful in the development of new drugs toinhibit 33 kDa(BP) cytotoxic activity. Inhibition of 33 kDa(BP)cytotoxicity can lower a patient's susceptibility to lung inflammationand enhance the patient's recovery rate. The discovery of 33 kDa(BP)therefor permits specific inhibitors to be developed to inhibitcytotoxicity in the airways of patients with lung diseases and to lowera patient's susceptibility to lung injury. I have discovered thatAnnexin I and/or 33 kDa(PLBP) can effectively inhibit the cytotoxicactivity of the Annexin I breakdown product 33 kDa (BP).

33 kDa(BP) and the ratio of 33 kDa(BP)/Annexin I in BAL fluid also canbe used in a diagnostic kit to diagnose or predict lung injury by usinga specific antibody to 33 kDa(BP) to detect the presence of 33 kDa(BP).

V. Analysis of Annexin I in Human Amniotic Fluid (AF)

I also have discovered that the presence of Annexin I and 33 kDa(BP) inamniotic fluid can be useful in diagnosing high risk pregnancies.

In four AF specimens from four patients with high risk pregnancies,Annexin I and 33 kDa(BP) were detected in two of the AF specimens,whereas two AF specimens contained no Annexin I but only 33 kDa(BP). Itis likely that these latter amniotic fluid specimens containedproteolytic enzymes which hydrolyzed Annexin I to yield the 33 kDa(BP).In addition, preliminary data showed that the breakdown of Annexin I inamniotic fluid from patients with high risk pregnancy was similar tothat in the BAL fluid from patients with lung inflammation. Elastaseinhibitor (PMSF) prevented Annexin I degradation in the presence ofelastase.

In addition, to using the analysis of the amount of Annexin I/33kDa(PLBP) in amniotic fluid as a means to predict premature delivery,the administration of Annexin I and/or 33 kDa(PLBP) into the amnioticfluid will help to prevent the infections, effects of cytokines andprostaglandins which are the major causes of premature delivery. Themechanism of the infection in amniotic fluid is similar to that in theairway.

The preferred pharmaceutical compositions, in addition to the Annexin I,the 33 kDa(PLBP) or mixtures thereof, will contain a source of calciumions. When intended for instillation into the lungs of an animal, theyalso will contain a surfactant or surface active agent.

The Annexin I and 33 kDa(PLBP) are natural proteins in the airway.Therefore, the side effects and toxicities of these proteins areexpected to be minimal. Both human Annexin I and 33 kDa(PLBP) can besynthesized or produced by genetic engineering techniques.

The preferred source of calcium ions is a soluble calcium salt, such ascalcium carbonate or calcium chloride. The amount of calcium ions in thecompositions will depend upon the amount of active ingredient. Thecalcium ion concentration can range from zero where enough endogenouscalcium is present to about 1 mM or more.

A preferred surfactant would be one that is known to be beneficial inthe treatment of lung disease, such as that sold under the trademarkSURVANTA by Ross Laboratories. Suitable surface active agents includeboth non-fluorinated surfactants and fluorinated surfactants known inthe art and disclosed, for example, in British Patent Nos. 837465 and994734 and U.S. Pat. No. 4,352,789.

Examples of other surfactants include:

Sorbitan trioleate,

Sorbitan mono-oleate,

Sorbitan monolaurate,

Polyoxyethylene (20) sorbitan monolaurate,

Polyoxyethylene (20) sorbitan mono-oleate,

Lecithins derived from natural sources

Oleyl polyoxyethylene ether,

Stearyl polyoxyethylene,

Lauryl polyoxyethylene ether, and

Oleyl polyoxyethylene ether.

The surfactants or surface active agents are generally present inamounts not exceeding 5 percent by weight of the total formulation. Theywill usually be present in the weight ratio 1:100 to 10:1 surface activeagent: active ingredient, but the surface active agent may exceed thisweight ratio in cases where the active ingredient concentration in theformulation is very low.

The particle size of the active ingredients should desirably be nogreater than 100 microns diameter. Preferably, the particle size of afinely-divided solid powder should for physiological reasons be lessthan 25 microns and preferably less than about 10 microns in diameter.The particle size for inhalation therapy should preferably be in therange 0.5 to 10 microns.

The concentration of the active ingredient depends upon the desireddosage, but it is generally in the range 0.01 to 5% by weight. Thedosage will usually be selected to bring the levels of the Annexin I or33 kDa(PLBP) at least up to the levels of those polypeptides found inthe lung or amniotic fluid of normal animals. It should be noted,however, that the dosage can be adjusted to any level which is toleratedwithout substantial adverse effect by the patient. Thus, dosages of from0.05 μg/kg of the animal's body weight up to 500 mg/kg or higher couldbe used if such high levels are not toxic and produce the desirableresult in the patient. The availability of an animal model for cysticfibrosis, mice homozygous for a disrupted CFTR gene, allows for thetesting of compositions containing the active ingredients in animalswithout undue experimentation.

A representative composition for instillation into the lungs of animalwould contain in 1 ml. about 0.02 mg of Annexin I or 33 kDa(PLBP); 0.5mg of calcium (5 mM) and 0.4 mg of surfactant phospholipids. It couldalso contain other diluents and ingredients and it would be packaged inan aerosol form not requiring a CFC propellant. A composition foraddition to amniotic fluid would not contain the surfactant.

From the foregoing, it will be apparent to those skilled in the art thatthis invention has wide and broad clinical applications for children andadults with cystic fibrosis, HIV/AIDS immune suppressed patients,neutropenic, post-operative, bed ridden and chronic obstructivepulmonary disease patients, as well as, infants with bronchopulmonarydysplasia and patients with septic shock or patients who have high riskpregnancies.

It will be apparent to those skilled in the art that a number ofmodifications and changes can be made without departing from the scopeof the invention. Therefore, it is intended that the invention belimited only by the claims.

What is claimed is:
 1. A method of treating an animal to prevent oralleviate the adverse effects of endotoxin in the lung, said methodcomprising administering into the airway of an animal a safe amount of33 kDa(PLBP) which is effective to prevent or alleviate the adverseeffects of endotoxin.
 2. A method of claim 1 in which a surfactant isadministered with the 33 kDa(PLBP) into the animal's airway.